Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 16;11(5):e0155548.
doi: 10.1371/journal.pone.0155548. eCollection 2016.

Novel Luminex Assay for Telomere Repeat Mass Does Not Show Well Position Effects Like qPCR

Muhammad G Kibriya  1 , Farzana Jasmine  1 , Shantanu Roy  1 , Habibul Ahsan  1 , Brandon L Pierce  1
Affiliations

Novel Luminex Assay for Telomere Repeat Mass Does Not Show Well Position Effects Like qPCR

Muhammad G Kibriya et al. PLoS One. .

Abstract

Telomere length is a potential biomarker of aging and risk for age-related diseases. For measurement of relative telomere repeat mass (TRM), qPCR is typically used primarily due to its low cost and low DNA input. But the position of the sample on a plate often impacts the qPCR-based TRM measurement. Recently we developed a novel, probe-based Luminex assay for TRM that requires ~50ng DNA and involves no DNA amplification. Here we report, for the first time, a comparison among TRM measurements obtained from (a) two singleplex qPCR assays (using two different primer sets), (b) a multiplex qPCR assay, and (c) our novel Luminex assay. Our comparison is focused on characterizing the effects of sample positioning on TRM measurement. For qPCR, DNA samples from two individuals (K and F) were placed in 48 wells of a 96-well plate. For each singleplex qPCR assay, we used two plates (one for Telomere and one for Reference gene). For the multiplex qPCR and the Luminex assay, the telomere and the reference genes were assayed from the same well. The coefficient of variation (CV) of the TRM for Luminex (7.2 to 8.4%) was consistently lower than singleplex qPCR (11.4 to 14.9%) and multiplex qPCR (19.7 to 24.3%). In all three qPCR assays the DNA samples in the left- and right-most columns showed significantly lower TRM than the samples towards the center, which was not the case for the Luminex assay (p = 0.83). For singleplex qPCR, 30.5% of the variation in TL was explained by column-to-column variation and 0.82 to 27.9% was explained by sample-to-sample variation. In contrast, only 5.8% of the variation in TRM for the Luminex assay was explained by column-to column variation and 50.4% was explained by sample-to-sample variation. Our novel Luminex assay for TRM had good precision and did not show the well position effects of the sample that were seen in all three of the qPCR assays that were tested.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Variation of Telomere products by column.
Column number is shown in x-axis and the quantity of Telomere product is shown in y-axis. CT-values of Telomere product (inversely proportional to PCR product quantity) from SP-qPCR-set1, SP-qPCR-set2 and MP-qPCR are shown in fig (A), (B) and (C) respectively. Quantity of Telomere product measured by Luminex assay is shown in (D).
Fig 2
Fig 2. Variation of reference gene products by column.
Column number is shown in x-axis and the quantity of reference gene product is shown in y-axis. CT-values of the Reference gene (inversely proportional to PCR product quantity) from SP-qPCR-set1, SP-qPCR-set2 and MP-qPCR are shown in fig (A), (B) and (C) respectively. Quantity of the Reference gene (ALK) product measured by Luminex assay is shown in (D).
Fig 3
Fig 3. Variation of Relative Telomere repeat mass measures from different assays by column.
Column number is shown on the x-axis and the TRM is shown on the y-axis. The qPCR-index measured by SP-qPCR-set1, SP-qPCR-set2 and MP-qPCR are shown in fig (A), (B) and (C) respectively. qPCR index was calculated as 2-deltaCT(test sample)/2-deltaCT(control sample) = 2-delta delta CT. Telomere Quantity Index (TQI) measured by Luminex assay is shown in (D). TQI was calculated as Telomere/ALK which is normalized for quantity of DNA in a well and it is relative to the “standard” DNA sample.
Fig 4
Fig 4. Variation of “single gene” measures from different assays by column or row position.
The CT values for the randomly selected single gene MTHFR and the reference gene 36B4 by column are shown in (A) and (B) respectively. The variation of relative abundance of single gene MTHFR (measured as MTHFR/36B4 ratio) by column and row is shown in (C) and (D) respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • Bovine telomere dynamics and the association between telomere length and productive lifespan.
    Seeker LA, Ilska JJ, Psifidi A, Wilbourn RV, Underwood SL, Fairlie J, Holland R, Froy H, Salvo-Chirnside E, Bagnall A, Whitelaw B, Coffey MP, Nussey DH, Banos G. Seeker LA, et al. Sci Rep. 2018 Aug 24;8(1):12748. doi: 10.1038/s41598-018-31185-z. Sci Rep. 2018. PMID: 30143784 Free PMC article.
  • The contribution of parent-to-offspring transmission of telomeres to the heritability of telomere length in humans.
    Delgado DA, Zhang C, Gleason K, Demanelis K, Chen LS, Gao J, Roy S, Shinkle J, Sabarinathan M, Argos M, Tong L, Ahmed A, Islam T, Rakibuz-Zaman M, Sarwar G, Shahriar H, Rahman M, Yunus M, Doherty JA, Jasmine F, Kibriya MG, Ahsan H, Pierce BL. Delgado DA, et al. Hum Genet. 2019 Jan;138(1):49-60. doi: 10.1007/s00439-018-1964-2. Epub 2018 Dec 10. Hum Genet. 2019. PMID: 30536049 Free PMC article.
  • The association between telomere length and mortality in Bangladesh.
    Dean SG, Zhang C, Gao J, Roy S, Shinkle J, Sabarinathan M, Argos M, Tong L, Ahmed A, Islam MT, Islam T, Rakibuz-Zaman M, Sarwar G, Shahriar H, Rahman M, Yunus M, Graziano JH, Chen LS, Jasmine F, Kibriya MG, Ahsan H, Pierce BL. Dean SG, et al. Aging (Albany NY). 2017 Jun 15;9(6):1537-1551. doi: 10.18632/aging.101246. Aging (Albany NY). 2017. PMID: 28630379 Free PMC article.
  • Determinants of telomere length across human tissues.
    Demanelis K, Jasmine F, Chen LS, Chernoff M, Tong L, Delgado D, Zhang C, Shinkle J, Sabarinathan M, Lin H, Ramirez E, Oliva M, Kim-Hellmuth S, Stranger BE, Lai TP, Aviv A, Ardlie KG, Aguet F, Ahsan H; GTEx Consortium; Doherty JA, Kibriya MG, Pierce BL. Demanelis K, et al. Science. 2020 Sep 11;369(6509):eaaz6876. doi: 10.1126/science.aaz6876. Science. 2020. PMID: 32913074 Free PMC article.
  • Urinary arsenic and relative telomere length in 5-7 year old children in Bangladesh.
    Farzan SF, Shahriar M, Kibriya MG, Jasmine F, Sarwar G, Slavkovic V, Graziano JH, Ahsan H, Argos M. Farzan SF, et al. Environ Int. 2021 Nov;156:106765. doi: 10.1016/j.envint.2021.106765. Epub 2021 Jul 15. Environ Int. 2021. PMID: 34273872 Free PMC article.
See all "Cited by" articles

References

    1. Sanders JL, Newman AB. Telomere length in epidemiology: a biomarker of aging, age-related disease, both, or neither? Epidemiol Rev; 2013; 35: 112–31. 10.1093/epirev/mxs008 - DOI - PMC - PubMed
    1. Blackburn EH. Structure and function of telomeres. Nature; 1991; 350: 569–73. - PubMed
    1. Blackburn EH. Switching and signaling at the telomere. Cell; 2001; 106: 661–73. - PubMed
    1. Lapham K, Kvale MN, Lin J, Connell S, Croen LA, Dispensa BP, et al. Automated Assay of Telomere Length Measurement and Informatics for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort. Genetics; 2015; 200: 1061–72. 10.1534/genetics.115.178624 - DOI - PMC - PubMed
    1. Hou L, Joyce BT, Gao T, Liu L, Zheng Y, Penedo FJ, et al. Blood Telomere Length Attrition and Cancer Development in the Normative Aging Study Cohort. EBioMedicine; 2015; 2: 591–6. 10.1016/j.ebiom.2015.04.008 - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources